It is now well established that in activated human neutrophils 1-0-alkyl-2-arachidonoyl-sn-glycero-3- phosphocholine (alkyl-2-AA-GPC) serves as a source of both platelet activating factor (PAF) and arachidonic acid (AA), which can be converted to bioactive eicosanoids. Dr. Wykle's laboratory has recently found evidence that in human neutrophils hydrolysis of AA from 1-0-alk-1-enyl-2- AA-sn-glycero-3-phosphoethanolamine (alkenyl-2-AA-GPE) by phospholipase A2 (PLA2) results in the accumulation of alkenyl-2-lyso-GPE which can trigger transfer of AA from alkyl-AA-GPC to the alkenyl-2-lyso-GPE, thus regenerating alkenyl-2-AA-GPE and forming alkyl-2-lyso-GPC (lyso PAF). The studies outlined are designed to assess the importance of this indirect route of AA release and PAF synthesis relative to that catalyzed by the direct action of PLA2 on alkyl-2-AA-GPC, which could release AA and lyso-PAF without the action of the transacylase. The transacylase will be characterized both in membrane preparations and as the purified enzyme, if attempts to isolate the enzyme are successful. The substrate specificity, kinetic parameters, protein properties, and control mechanisms of the transacylase will be examined. In the postulated transacylase-dependent route of AA release and PAF synthesis, the action of PLA2 on alkenyl-2-AA-GPE plays a central role in triggering the pathway. Studies will be carried out to examine the hydrolysis of exogenous and membrane-bound alkenyl-AA-GPE by PLA2 in both isolated subcellular fractions and in permeabilized cells. These studies will provide information on the nature of the PLA2 responsible for the release of AA and synthesis of PAF and will elucidate the control mechanisms responsible for activating the enzyme upon stimulation. Our preliminary evidence suggests that the alkyl-2-AA-GPC and alkenyl-2-AA- GPE of the specific granules may play an important role as a source of eicosanoids and PAF in stimulated neutrophils. The distribution f the transacylase and PLA2, as well as their substrates and products, will be closely examined in the granules and membranes of resting and stimulated neutrophils. If these findings verify that the specific granules play an important role in the synthesis of the lipid mediators, the possible role of fusion of specific granules and plasma membrane in activation of the synthetic pathways will be examined. The studies will employ a number of organically synthesized phospholipid analogs as possible affinity ligands for purifying the transacylase and as potential inhibitors of the PLA2 and transacylase.
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